Rapid quantification of planktonic ciliates: comparison of improved live counting with other methods

THE FOLLOWING EFFICIENT AND QUANTITATIVELY VALID METHOD TO FILTER CONCENTRATE AND COUNT LIVE PLANKTONIC CILIATES WAS DEVELOPED AND COMPARED WITH OTHER TREATMENTS: unconcentrated (raw) samples and centrifuged samples were counted live, and the effects of five different fixatives (HgCl(2), Lugol's iodine, formaldehyde, glutaraldehyde, and Champy-DaFano) on the counts were monitored. Samples originated from a eutrophic mountain lake (Lake Aydat, near Clermont-Ferrand, France). Overall, live filtered counts were similar to counts of raw samples, but they were significantly higher (2 to 2.3 fold, P < 0.05) by analysis of variance than counts from centrifuged samples. Nevertheless, some taxa, i.e., Halteria and Loxodes spp., were sensitive to filtration. The live filtered counts were also comparable to counts of raw HgCl(2)-fixed and settled samples. HgCl(2) and Lugol fixation consistently gave the highest total counts, while significantly lower counts were always obtained with Champy-DaFano-fixed samples. Losses due to fixation were insignificant for raw samples but were substantial and statistically significant in concentrated samples (15% after filtration and 71% after centrifugation, compared with counts from the corresponding live samples). Live counting of passively filter-concentrated ciliates has many advantages over other methods. It is two to four times quicker and more efficient. Ciliates are recognized with certainty, more species are identified, and enumeration of dead organisms (e.g., tintinnid loricas) is avoided. It should be recommended as a quantitatively valid alternative to classical methods for assessing planktonic ciliate populations.     

A Human In Vitro Whole Blood Assay to Predict the Systemic Cytokine Response to Therapeutic Oligonucleotides Including siRNA

Therapeutic oligonucleotides including siRNA and immunostimulatory ligands of Toll-like receptors (TLR) or RIG-I like helicases (RLH) are a promising novel class of drugs. They are in clinical development for a broad spectrum of applications, e.g. as adjuvants in vaccines and for the immunotherapy of cancer. Species-specific immune activation leading to cytokine release is characteristic for therapeutic oligonucleotides either as an unwanted side effect or intended pharmacology. Reliable in vitro tests designed for therapeutic oligonucleotides are therefore urgently needed in order to predict clinical efficacy and to prevent unexpected harmful effects in clinical development. To serve this purpose, we here established a human whole blood assay (WBA) that is fast and easy to perform. Its response to synthetic TLR ligands (R848: TLR7/8, LPS: TLR4) was on a comparable threshold to the more time consuming peripheral blood mononuclear cell (PBMC) based assay. By contrast, the type I IFN profile provoked by intravenous CpG-DNA (TLR9 ligand) in humans in vivo was more precisely replicated in the WBA than in stimulated PBMC. Since Heparin and EDTA, but not Hirudin, displaced oligonucleotides from their delivery agent, only Hirudin qualified as the anticoagulant to be used in the WBA. The Hirudin WBA exhibited a similar capacity as the PBMC assay to distinguish between TLR7-activating and modified non-stimulatory siRNA sequences. RNA-based immunoactivating TLR7/8- and RIG-I-ligands induced substantial amounts of IFN-α in the Hirudin-WBA dependent on delivery agent used. In conclusion, we present a human Hirudin WBA to determine therapeutic oligonucleotide-induced cytokine release during preclinical development that can readily be performed and offers a close reflection of human cytokine response in vivo.     

Human B cell lines can be triggered to secrete an interleukin 2-like molecule

To determine whether human B cells can be triggered to secrete interleukin 2 (IL-2), 19 tumor cell lines derived from patients with undifferentiated lymphomas of Burkitt's and non-Burkitt's types and 6 normal lymphoblastoid cell lines were tested. Cells were grown in the presence or absence of the new tumor promoter teleocidin, and culture supernatants were assayed for IL-2 activity using the standard CTLL-2 assay. Teleocidin (10 ng/ml) triggered IL-2 secretion in 7/8 (87%) EBV-negative lymphoma cell lines of American origin and in 6/6 (100%) normal lymphoblastoid cell lines, but in only 1/6 (16%) EBV-positive tumor cell lines of American origin. Teleocidin had no effect on 5/5 (0%) African Burkitt's cell lines. IL-2 secretion was not detected in control supernatants. IL-2 secretion correlated with the induction of IgM secretion and was linked to both EBV status and karyotype. The following similarities in the functional biological characteristics of T cell and B cell IL-2 suggest that B cell IL-2 is not a factor which mimics IL-2 activity in the CTLL-2 assay: (i) neutralization of IL-2 by anti-IL-2 monoclonal antibody (DMS-1); (ii) elution of IL-2 following its adsorption to CTLL-2 cells; (iii) determination of the MW of IL-2 by SDS-PAGE and Western blot analysis; and (iv) ability of B cell IL-2 to support T cell proliferation and blocking of this activity by anti-tac monoclonal antibody. cDNA probes for T cell IL-2, however, did not detect IL-2 mRNA in B cells. The cell lines were also found to constitutively express IL-2 receptors detected by anti-tac monoclonal antibody, and to secrete soluble IL-2 receptors measured by ELISA. Our results imply that under certain circumstances, B cells can be triggered to secrete IL-2 or an IL-2-like molecule and thus influence T cell activation and proliferation.     

Differentiation of somatic embryos from protoplasts isolated from embryogenic suspension cultures of Abies alba L.

Embryogenic suspension cultures of Abies alba were established using an embryogenic suspensor mass culture originating from the zygotic embryo in immature seed explants (Schuller et al. 1989). Protoplasts were isolated from the suspension material. The protoplasts were immobilized in alginate layers in order to follow the development of single protoplasts. During the first days of protoplast culture a modified Kao and Michayluk (1975) medium proved to be necessary for subsequent divisions. The formation of proembryos succeeded within 2–3 weeks when subcultured with a modified Schenk and Hildebrandt (1972) liquid medium. Light, enhanced sugar concentration, and the addition of abscisic acid led to the formation of slightly green torpedo-shaped somatic embryos after 6–8 weeks from protoplast isolation.Abbreviations ABA abscisic acid - BAP N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - ESM Embryonal suspensor mass (Gupta and Durzan 1986) - KM Kao and Michayluk (1975) - LP (von Arnold and Eriksson 1977) - MES 2-(N-morpholino)ethane-sulfonic acid - NAA 1-naphthalene-acetic acid (sodium salt) - PVP polyvinylpyrrolidone - SH Schenk and Hildebrandt (1972) - Tween 80 polyoxyethylene-sorbitan-monooleate     

Comparative immunochemical study of ribosomal proteins from various mammalian cells by two-dimensional immunoelectrophoresis

Summary Proteins isolated from ribosomal subunits of various mammalian cells were analysed comparatively by two different methods: a two-dimensional polyacrylamide gel electrophoresis system and a recently described two-dimensional immunoelectrophoresis technique. For this purpose, antisera were raised in rabbits against the total mixture of ribosomal proteins from murine cells. These sera were characterized by ring-test, double immunodiffusion and two-dimensional immunoelectrophoresis. They were shown to contain antibodies to a large number of ribosomal proteins. Immunoelectrophoretic analysis of 60S and 40S subunit proteins from rabbit, lamb, canine and human cells using anti-murine sera revealed a striking conservation of their antigenic properties. These results corroborated those obtained by two-dimensional polyacrylamide gel electrophoresis.     

Genomic deletion and promoter methylation status of Hypermethylated in Cancer 1 (HIC1) in mantle cell lymphoma

Mantle cell lymphomas (MCL), characterized by the t(11;14)(q13;q32), frequently carry secondary genetic alterations such as deletions in chromosome 17p involving the TP53 locus. Given that the association between TP53-deletions and concurrent mutations of the remaining allele is weak and based on our recent report that the Hypermethylated in Cancer 1 (HIC1) gene, that is located telomeric to the TP53 gene, may be targeted by deletions in 17p in diffuse large B-cell lymphoma (DLBCL), we investigated whether HIC1 inactivations might also occur in MCL. Monoallelic deletions of the TP53 locus were detected in 18 out of 59 MCL (31%), while overexpression of p53 protein occurred in only 8 out of 18 of these MCL (44%). In TP53-deleted MCL, the HIC1 gene locus was co-deleted in 11 out of 18 cases (61%). However, neither TP53 nor HIC1 deletions did affect survival of MCL patients. In most analyzed cases, no hypermethylation of the HIC1 exon 1A promoter was observed (17 out of 20, 85%). However, in MCL cell lines without HIC1-hypermethylation, the mRNA expression levels of HIC1 were nevertheless significantly reduced, when compared to reactive lymph node specimens, pointing to the occurrence of mechanisms other than epigenetic or genetic events for the inactivation of HIC1 in this entity.     

Effect of insulin pump therapy on blood pressure and the renin-angiotensin system of diabetic rats

Insulin therapy, administered by continuous subcutaneous infusion with osmotic pumps over a 28 day period at doses of 2.5 and 5.0 units/day, resulted in a statistically significant increase in body weight of diabetic rats. The concentration of blood glucose was reduced by 68% to 109 mg/dl blood sugar by the higher dose of insulin and only partial control of diabetes was achieved by the lower dose (185 mg/dl blood sugar, -39%). Blood pressure was normalized by both doses of insulin. Elevated serum angiotensin converting enzyme activity and plasma renin activity, expressed as generated angiotensin I, were unaffected by the lower dose of insulin, but were reduced by 26% and 40%, respectively at the higher dose. These data suggest that elevated serum ACE and plasma renin activity, commonly found in the streptozotocin-diabetic rat, may not be primarily responsible for hypertension in this model.     

In situ identification of intracellular bacteria related to Paenibacillus spp. in the mycelium of the ectomycorrhizal fungus Laccaria bicolor S238N

Bacterial proliferations have recurrently been observed for the past 15 years in fermentor cultures of the ectomycorrhizal fungus Laccaria bicolor S238N, suggesting the presence of cryptic bacteria in the collection culture of this fungus. In this study, intracellular bacteria were detected by fluorescence in situ hybridization in combination with confocal laser scanning microscopy in several collection subcultures of L. bicolor S238N. They were small (0.5 micro m in diameter), rare, and heterogeneously distributed in the mycelium and were identified as Paenibacillus spp. by using a 16S rRNA-directed oligonucleotide probe initially designed for bacteria isolated from a fermentor culture of L. bicolor S238N.